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Keywords:

  • Ti–Nb;
  • biomedical;
  • chemical treatment;
  • XPS;
  • mesenchymal stromal cells

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. EXPERIMENTAL
  5. RESULTS AND DISCUSSION
  6. SUMMARY AND CONCLUSIONS
  7. Acknowledgments
  8. REFERENCES

Samples of low modulus beta-type Ti40Nb and cp2-Ti were chemically treated with 98% H2SO4 + 30% H2O2 (vol. ratio 1:1) solution. Surface analytical studies conducted with HR-SEM, AFM, and XPS identified a characteristic nanoroughness of the alloy surface related with a network of nanopits of ∼25 nm diameter. This is very similar to that obtained for cp2-Ti. The treatment enhances the oxide layer growth compared to mechanically ground states and causes a strong enrichment of Nb2O5 relative to TiO2 on the alloy surface. The in vitro analyses clearly indicated that the chemical treatment accelerates the adhesion and spreading of human mesenchymal stromal cells (hMSC), increases the metabolic activity, and the enzyme activity of tissue non-specific alkaline phosphatase (TNAP). Surface structures which were generated mimic the cytoplasmic projections of the cells on the nanoscale. Those effects are more pronounced for the Ti40Nb alloy than for cp2-Ti. The relation between alloy surface topography and chemistry and cell functions is discussed. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 102B: 31–41, 2014.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. EXPERIMENTAL
  5. RESULTS AND DISCUSSION
  6. SUMMARY AND CONCLUSIONS
  7. Acknowledgments
  8. REFERENCES

A world-wide growing population of aged people and the related rising problem of age-induced diseases of bones like osteoporosis increase the demands on material properties used for human orthopedic implants towards better mechanical and biological compatibility and longevity.2009, 2008 In the class of non-degradable implant materials for hard tissue replacements those based on Ti are most promising with respect to their properties, workability and costs.2009, 2003 Currently, cp(2)-Ti (α-type) and Ti6Al4V (α+β-type) are widely established in orthopedic surgery and are still the main target of fundamental research. Strong efforts are devoted to further improvement of their biocompatibility which is determined by the interactions at the bone tissue/implant interface. The implant surface state comprising topography and roughness, composition and surface energy controls these interactions.2009 Therefore, numerous approaches for tailoring Ti-based implant surfaces have been proposed based on mechanical, chemical or physical surface modification techniques and those are comprehensively reviewed, for example, in Refs. 2004 and 2008. While many of these treatments yield distinct micrometer-sized topographies, it has been already recognized that nanometer-sized surface modifications would better scale with cell functions and therefore, further increase the biofunctionality.2011, 2009, 2006 In recent studies, a novel technique for nanoroughening of cp-Ti surfaces by chemical treatment in an oxidative H2SO4/H2O2 electrolyte (piranha solution) was proposed which comprises etching of multiple nanopits forming a “nanoporous” or “nanorough” layer and enhanced TiO2 formation.2009, 2006 A high capability of these surface states for selectively influencing protein adsorption2010 and bone cell behavior was evidenced, the latter by an upregulation of the early expression of bone sialoprotein II and osteopontin in osteogenic cell cultures and an enhancement of contact osteogenesis.2004, 2007

New developments in the field of Ti-based implant materials are β-type alloys with superior properties compared to cp-Ti and Ti6Al4V.2009, 2003, 1998, 2007, 2011 The key differentiator is the body-centered cubic microstructure which causes an improved workability and remarkably lower elastic moduli. Especially the decreased Young's modulus will reduce the so-called stress shielding effects.1983, 2008, 1987 and therefore cause diminished pathologic bone resorption. The typically higher strengths compared to other titanium alloys can lower traumatic and fatigue fracture rates, respectively. Moreover, these new alloys comprise only β-stabilizing elements like Nb, Ta, Zr, Mo, or Sn with very low cytotoxicity, which at the same time cause a high corrosion resistance related with very low metal release rates. This implies significantly improved tissue reactions and longevity of the implant material.

A very promising alloy system is Ti-Nb, for which at 40–45 Wt % Nb a minimum of the elastic modulus of 60–62 GPa can be obtained. This is only the half of the value of established Ti-based materials. By suitable thermo-mechanical processing and micro-alloying of cast samples, further modulus reduction to 40–50 GPa and high tensile strengths of >1 GPa can be obtained. These properties are mainly related with the formation of a pure metastable β-type phase and simultaneous suppression of competing ω- and α″-phases.2005, 1988 The high Nb-content triggers a spontaneous surface passivation in artificial body fluids with very strong barrier effect against pitting corrosion.2006, 2011, 2013. However, the excellent chemical stability brings new challenges on chemical surface modification treatments which are also, in this case, indispensible for optimum interfacial bone/implant interactions. For Ti45Nb, the effect of a few treatments was already investigated, that is, electropolishing, anodic and thermal oxidation,2006 electrochemical alkali-treatment,2003 or the chemical deposition of self-assembled organic monolayers.2005, 2007 We have shown in a recent study that acid etching procedures established for Ti and Ti6Al4V are not sufficient for Ti45Nb and that more severe conditions are needed. First tests revealed the effective applicability of the H2SO4/H2O2 treatment for nanoroughening the alloy surface without altering the corrosion resistance.2013

In this article, a detailed surface analytical characterization of H2SO4/H2O2 treated surface states of Ti40Nb alloy samples in relation to polished reference states is presented. It will be demonstrated that the chemical treatment accelerates the adhesion and spreading of human mesenchymal stromal cells (hMSC), increases the metabolic activity and the enzyme activity of tissue non-specific alkaline phosphatase (TNAP) and that this effect is even more pronounced for the Ti40Nb alloy than for cp2-Ti. The relation between alloy surface topography and chemistry and cell functions will be discussed, as well.

EXPERIMENTAL

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. EXPERIMENTAL
  5. RESULTS AND DISCUSSION
  6. SUMMARY AND CONCLUSIONS
  7. Acknowledgments
  8. REFERENCES

Alloy preparation, microstructure analyses, and surface treatment

Ti40Nb (Wt %) ingots were prepared by arc-melting high purity Ti and Nb in an Ar atmosphere and re-melting them several times for homogenization. These pre-alloys were subsequently melted by induction heating and cold crucible cast with an overpressure of 500 mbar in a water-cooled copper mould with a diameter of 10 mm and length of 100 mm. The rods were subjected to a homogenization treatment at 1000°C for 24 h under highly purified argon atmosphere. Slices of 2–3 mm thickness were cut from these rods for the following experiments. The alloy composition was checked by inductively coupled plasma optical emission spectroscopy (ICP-OES) and was found to coincide with the nominal one in the error limits of the method.

Microstructural characterization of Ti40Nb samples was performed by X-ray diffraction (XRD) using Mo Kα radiation with a STOE Stadi P instrument in transmission mode and by scanning electron microscopy (SEM) with a Gemini Leo 1530 instrument. To reveal its grain structure, a sample for SEM was sputtered in a glow discharge Ar plasma.2013

In this study, the nature and the cell biological effect of a chemically treated surface should be compared with that of an application-relevant mechanically ground state. Therefore, in a standard procedure, disc-shaped alloy and Ti samples were ground with SiC emery paper up to grit P1200. As reference material for the cell biological tests, grade 2 commercial pure titanium (cp2-Ti) supplied by ARA-T advance GmbH (Germany) was used. According to ASTM F67 cp2-Ti is suitable for biomedical application and generally used for surgical implants. Disc-shaped samples with the same diameter were manufactured by electro-discharge machining and ground consistently in the same way as the alloy samples.

Subsequently, a part of these samples was additionally treated in 98% H2SO4 + 30% H2O2 (vol. ratio 1:1; piranha solution), whereby Ti40Nb samples were exposed for 24 h and cp2-Ti samples only for 8 h. The exposure times were chosen in result of pre-tests revealing in first microscopic assessments nearly similar surface states for both materials. The treated samples were then rinsed several times with distilled water to remove all contaminants of the aggressive piranha solution and dried in air.

Those samples were subjected to standard topographic surface analysis by means of high resolution scanning electron microscopy HR-SEM (Gemini Leo 1530) and profilometry (MicroProf FRT GmbH),2013 to hydrogen analysis by hot gas extraction (Horiba) and to cell biological tests (a set of eight samples per material and surface state).

For more detailed surface analytical characterizations of the chemically treated alloy surfaces on the nanoscale by means of high resolution SEM, atomic force microscopy (AFM), and X-ray photoelectron spectroscopy (XPS), very smooth sample surfaces on the microscale are indispensible for the achievement of high precision and reliability of the results. Therefore, for these analyses, the samples were further fine-polished with OP-S suspension (a water-based suspension of SiO2 nanoparticles of 40 nm size) and carefully rinsed with water and alcohol prior to the H2SO4/H2O2 exposure.

Analysis of the surface nanotopography with HR-SEM and AFM

High Resolution SEM analysis (SE mode; FE-REM Ultra Plus, Fa. Zeiss) was employed to characterize the surface state of chemically treated cp2-Ti and Ti40Nb samples at the submicron-scale. SEM images were taken with acceleration voltages of 5 and 10 kV, respectively. The high resolution images revealing a continuous pattern of nanopits were subjected to an image analyzer (QTM550, Fa. Leica) in order to determine the size distribution of the pits. This was done by measuring pit areas and calculating the diameters of area-equivalent circles. For segmentation of the objects, a “Watershed algorithm”1998 performed in the SEM grey value image after a slight smoothing was identified to be the most reliable procedure when assuming a continuous cell wall dimension of 1 pixel (about 1 nm in the chosen magnification). The final diameter distribution curves were averaged from five images and were fitted with a log-normal distribution function.

The topography of the samples was imaged in the tapping mode with a DI 3100 atomic force microscope using super sharp silicon tips (SSS-MFMR, Nanosensors) with a guaranteed tip curvature of less than 15 nm. Two samples of each surface condition with typical areas of 1 µm × 1 µm were analyzed.

Analysis of surface chemistry with XPS and contact angle analysis

X-ray photoelectron spectroscopy (XPS) was used to characterize the chemical surface state of Ti40Nb samples. These measurements were carried out with a PHI 5600 CI (Physical Electronics) spectrometer which is equipped with a hemispherical analyzer running at typical pass energies of 90 eV for survey and of 29 eV for detailed spectra, respectively. At least two samples of each surface state were analyzed. The analysis area was around 800 µm in diameter. Monochromatic Al Kα excitation (350 W) was used at both 45° X-ray impact and electron emission angle with respect to the sample surface.

The estimation of oxide layer thickness was performed on the basis of the ratio of the measured Nb and Ti oxide and elemental signals originating from oxide layer and bulk, respectively. The calculation was done by the usual algorithm of exponential signal decay with an algorithm also used for multiple-angle XPS.2004 The necessary electron attenuation lengths were determined by an algorithm of Cumpson and Seah1997 taking into account a density of the polycrystalline Ti40Nb bulk material of 5.5 g/cm3 and value of 5.0 g/cm3 for the surface oxide. Separation of the spectra of the different chemical species was done by peak fitting of the measured using the PHI-MultiPak software.

The assessment of the wettability of the different surface states was done by means of dynamic contact angle measurements. The measurements were carried out with a OCA 35L device (Dataphysics) using purified distilled water at 23°C. The drops had an initial volume of 15 µL and were expanded with 0.1 µL/s. For the evaluation of the wettability only the advancing angle was considered. Each of the shown contact angles is a mean value of six samples belonging to the same surface state.

Analysis of hMSC response in vitro

For isolation of hMSC, bone marrow aspirates were collected from three bone marrow donors (males, average age 35 ± 4 years) at the Dresden Bone Marrow Transplantation Centre of the University Hospital Carl Gustav Carus. The study was approved by the local ethics commission (approval No. EK71022010; February 12 2010). The donors were informed and gave their approval. MSC were isolated and cultures according to Oswald et al.2004 hMSC of the 2.-4. passage were exposed to the different surfaces. 5,000 hMSC/cm² in DMEM with 10% fetal calf serum were placed in a 80 µL/cm² droplet directly onto the surface of the samples; 2 h after seeding culture medium was filled up until 0.5 mL/cm2. At day 4 after seeding, the medium was supplemented with 10 nM dexamethasone, 10 mM β-glycerophosphate, and 300 µM ascorbic acid to induce osteogenic differentiation.

2 h after seeding on the different samples, the cytoskeletal rearrangement and focal adhesion contact formation were monitored by fluorescence staining. Cells were washed with PBS and fixed with 4% paraformaldehyde (w/v;Sigma, Taufkirchen, Germany) in phosphate buffered saline (PBS) for 10 min. After permeabilization with 0.1% Triton X-100 (Sigma) in PBS for 20 min, non-specific binding sites were blocked with 1% bovine serum albumin (w/v; Sigma) in PBS containing 0.05% Tween-20 (Sigma). The cells were incubated with 64 µg of mouse anti-vinculin-IgG (HVin I clone)/mL (Sigma) PBS containing 1% bovine serum albumin and 0.05% Tween-20 for 1 h. After washing with PBS, cells were incubated with blocking buffer for 10 min again. Secondary antibody (10 µg AlexaFluor-568 goat anti-mouse-IgG/mL PBS containing 1% bovine serum albumin and 0.05% Tween 20) and 5 U AlexaFluor-488 phalloidin/mL (both Invitrogen, Karlsruhe, Germany) of PBS containing 1% bovine serum albumin and 0.05% Tween-20 were used for 1 h incubation at 25°C. Subsequently, 0.2 µg 4′,6-diamidino-2-phenylindole (DAPI)/mL (Sigma) PBS for nuclei staining was applied for 15 min at 25°C. After staining, the cells were embedded in Mowiol 4-88 (Sigma) and visualized using an AxioPhot fluorescence microscope (Carl Zeiss, Oberkochen, Germany). The fluorescence signals were detected with the following filters: excitation 546 nm and emission 590 nm for AlexaFluor-568, excitation 450–490 nm, and emission 515–565 nm for AlexaFluor-488, excitation 365 nm and emission 420 nm for DAPI. Digital images were obtained with an AxioCam MRm camera (Carl Zeiss) using AxioVision software release 4.6 (Carl Zeiss).

For SEM imaging (Gemini Leo 1530 instrument), hMSC were fixed 2 and 24 h after seeding with 4% paraformaldehyde (w/v) in PBS for 10 min. Afterwards the cells were drained in an ethanol series and following critical point dried. Electric conductivity was established by the physical vapor deposition of a thin gold coating.

Metabolic activity of hMSC was determined 24 h after seeding by the MTS assay (Cell Titer96 AQueous One Solution Proliferation Assay; Promega, Mannheim, Germany). 10% of MTS dye solution in DMEM was added. After 2 h of incubation at 37°C in a humidified CO2 incubator, 100 µL of medium was transferred into a 96-well plate and the absorbance of the formed MTS formazan dye was measured photometrically at 490/655 nm.

For determination of the catalytic activity of TNAP at day 11 after seeding, cells were washed with PBS and lysed in 1.5 M Tris–HCl, pH 10, containing 1 mM ZnCl2, 1 mM MgCl2, and 1% Triton X-100 (TNAP lysis buffer). After 10 min of incubation at 4°C, the lysates were harvested and clarified by centrifugation at 20,000g at 4°C for 30 min. Aliquots of the lysates were incubated with 3.7 mM 4-nitrophenylphosphate in 100 mM diethanolamine, pH 9.8, containing 0.1 % Triton X-100 at 37°C for 30 min. The reaction was stopped with 100 mM NaOH. Released 4-nitrophenolate was determined photometrically at 405 nm. Appropriate lysates were used for protein determination with RotiQuant protein assay (Roth GmbH, Karlsruhe, Germany) with bovine serum albumin as a standard. Specific catalytic activity of TNAP was calculated with a p-nitrophenolate calibration curve and the protein concentration of the lysate.

All presented data were derived from three independent experiments with three independent samples/hMSC donors (n = 3). The results are presented as mean ± standard error of the mean. Statistical significance was analyzed with two-way-ANOVA using Bonferroni's post-test.

RESULTS AND DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. EXPERIMENTAL
  5. RESULTS AND DISCUSSION
  6. SUMMARY AND CONCLUSIONS
  7. Acknowledgments
  8. REFERENCES

Microstructure characterization and surface state analyses

For microstructure characterization, homogenized Ti40Nb samples were investigated with XRD and SEM. In the diffraction pattern in Figure 1, only reflections corresponding to the β-(Ti,Nb) phase are detectable whereby no indications for ω- or α″-phases, which may occur as competing minor phases, are given. The inset in Figure 1 shows a SEM image of the sputtered cross sectional area revealing a typical structure of nearly equi-axed micrograins with sizes from a few tens to a few hundreds of micrometers and with different orientations as can be derived from the different sputter depths.

image

Figure 1. XRD pattern of a polished cross sectional area of a cast and homogenized Ti40Nb rod sample; inset: SEM image afterglow discharge sputtering.

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Figure 2 summarizes comparatively SEM images of different surface states of cp2-Ti (left) and Ti40Nb (right). In the mechanically ground state (a and b), both materials exhibit a similar surface morphology, that is, typical features are elongated grinding groves of micrometer dimension corresponding to the abrasive action of the SiC grains with mean size of ∼15 µm on the emery paper. For further surface analytical studies, the surfaces were fine-polished with OP-S suspension and subsequently treated with the H2SO4/H2O2 solution. In a series of pre-tests samples of both materials have been immersed in this solution for different durations up to 24 h and the surface morphology was evaluated [Figure (c–h)]. The cp2-Ti is readily reactive in this environment and the surface attack occurs at two levels. On the microscale [Figure 2(c)], preferential dissolution of grain boundary regions and of selected sites inside the grains, certainly related with surface defects like dislocation lines intersecting the surface, scratches etc., was observed already after short immersion times. In contrast, on the nanoscale multiple evolution of nanopits occurs. Already after 2 h of immersion a pronounced nanoroughening of the cp2 Ti surface can be observed [Figure 2(e)] as a preliminary stage of nanopitting. However, this process is more gradual, that is, longer immersion time is needed to achieve a homogeneous nanoroughening of the surface. Therefore, in order to find a compromise between limited micro-scale attack and complete nanoscale roughening for further tests with cp2-Ti, an immersion time of 8 h was determined [Figure 2(g)].

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Figure 2. SEM micrographs of surfaces of cp2-Ti (left) and Ti40Nb (right): (a,b) mechanically ground with SiC paper until grit 1200; (c–h) chemically treated with 98% H2SO4/30% H2O2 (vol. ratio 1:1, “piranha solution”).

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In comparison, the β-type Ti40Nb alloy is much less reactive in the piranha solution as can be seen in Figure 2(f). The surface of Ti40Nb is hardly affected after 2 h of etching which can be mainly attributed to the inhibiting effect of niobium—as already previously discussed.2013 Even after maximum exposure time of 24 h only weak corrosion attack is observed on the microscale [Figure 2(d)] at a few selected surface defects. Similar as for cp2-Ti, multiple pit formation on the nanoscale is possible, but much longer reaction times are needed for the evolution of a complete nanoroughened alloy surface [Figure 2(h)].

Figure 2(g) and (h) shows quite homogeneous nanoscale surface states for both materials were subsequently used for further topographic characterization. Results of SEM image analyses are shown in Figure 3 in terms of diameter distribution curves for assumed circular pits. Only marginal differences in the nanopit diameters were determined, that is, a mean diameter of 29.4 ± 0.4 nm (distribution width 14.4 ± 0.7 nm) for cp2-Ti and an only slightly lower value of 25.4 ± 0.4 nm (distribution width 12.2 ± 1.1 nm) for Ti40Nb were extracted. The value for cp2-Ti is slightly higher than that reported by Yi et al.,1998that is 22 ± 7 nm, who exposed the samples for only 2 h and employed another image analyzer software.

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Figure 3. Diameter distribution of nanopits on chemically treated cp2-Ti and Ti40Nb surfaces determined with SEM image analysis [Figure 2(g,h)] (Watershed algorithm).

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Further characterization of the chemically treated surface states was done by means of AFM. Figure 4 shows typical topographic images for cp2-Ti and Ti40Nb and corresponding line scans. In agreement with the SEM images, the cp2-Ti shows a rougher surface than Ti40Nb. Nevertheless, the structure of the nanopits looks different which can be attributed to the nominal curvature of the ultrasharp AFM tip. From the line scans a sequence of 2–3 minima per 100 nm length can be derived which correlates with the mean density of nano-pits observed by high resolution SEM. Also, depths of ∼15 to 17 nm for cp2-Ti and ∼12 to 15 nm for Ti40Nb can be estimated. Together with results of SEM diameter distribution analyses this indicates a nearly semi-spherical shape of the nanopits rather than a flat hollow-shape on both material surfaces.

image

Figure 4. AFM images (1 µm x 1µm) and corresponding line-scans of chemically treated cp2-Ti and Ti40Nb surfaces. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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In summary, those surface morphological and topographic studies revealed that by chemical treatment of Ti40Nb with H2SO4/H2O2 solution a nano-roughness similar to that of cp2-Ti surfaces can be attained. It must be emphasized that for cell biological studies the nano-roughness was superimposed with the microscopic pattern of the mechanically ground state.

The effect of the piranha treatment on the chemical state of the Ti40Nb alloy surface was analyzed by means of XPS. Figure 5 summarizes the detailed high resolution spectra of the alloying elements and of oxygen with peak fitting results for the different observed chemical states. The peak identification was done based on several literature sources.2007, 1993, 1999, 2008, 2005

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Figure 5. XPS analysis of mechanically polished and chemically treated Ti40Nb surfaces; measured and fitted core level spectra of Ti 2p, Nb 3d, and O 1s. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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In the mechanically polished state, the Ti 2p core level spectrum reveals the presence of a variety of valence states. Most pronounced is the doublet with peaks at 459.0 eV and at 464.7 eV which are related with the Ti4+ state of TiO2. Also clearly visible are the peaks at 453.8 eV and 460 eV related with Ti(0)metallic state. Moreover, indications for the existence of lower valence oxide states are given, that is, of Ti3+ like in Ti2O3 as reflected with a peak at 457.6 eV (and at 462.6 eV). In addition, the presence of a Ti2+ state like in TiO may be considered ascribed to weak reflections at 455.8 eV and 461.0 eV. From the Nb 3d core level spectrum also multiple valence states can be derived. The Nb5+ state with a doublet at 207.4 eV and 210.1 eV coexists with Nb(0)metallic related with peaks at 202.0 eV and 204.8 eV. Also in this case, presence of lower valence states has to be considered as expressed with peaks at 203.6 eV and 206.1 eV which can be related with Nb4+ (NbO2) or possibly Nb2+ (NbO). In relation to this, the O 1s spectrum of the mechanically ground state reveals a main peak at 530.5 eV which can be well ascribed to the O2− state mainly in bonding states of TiO2 and Nb2O5. Additionally, a characteristic shoulder occurs in the spectrum which can be fitted with peaks at 532 eV and 533.5 eV that are attributable to OH- bondings and H2O (adsorbed), respectively. Altogether, this spectra analysis demonstrates that on freshly polished Ti40Nb surfaces natural passive films grow under participation of both alloying elements. Main contributions of highest valence states Ti4+ and Nb5+ suggest the predominant coexistence of TiO2 and Nb2O5, but also some evidence for lower valence oxide states is given for both elements. The naturally formed oxide films are very thin—as obvious from the significant contribution of peaks of the metallic Nb and Ti states in the spectra. The estimation of the oxide film thickness based on the ratio of total metallic and oxide peak areas of both Ti and Nb yielded very low values of 2 nm. However, with respect to observations for other Ti-based alloys, for examplem Ti6Al7Nb [35], during long-term exposure up to one year under environmental conditions a very gradual thickness growth in the level <5 nm with slight alteration of film composition may be predicted.

After chemical treatment in H2SO4/H2O2 solution the core level spectra of the alloying elements still comprise the peaks of the discussed valence states but with strongly changed ratios. A significant enhancement of the peaks of the Ti4+ and Nb5+ states and simultaneous a distinct reduction of the peaks of the metallic and lower oxide states are indicative for a significant layer growth. Calculations of the oxide layer thickness yielded a mean value of 4.9 nm, that is, more than twice as high as that for the polished state. In accordance with the thickness growth of the oxide layer in the O 1s spectrum the O2− peak is enhanced relative to the shoulder. Furthermore, from a direct comparison of the change of the oxide/metal peak area ratios for Ti and Nb before and after the chemical treatment it became obvious that Nb-oxide species are enriched in the surface layer. The increase in the oxide/metal ratio of Nb was about 2.3 times of that for Ti—as it is obvious from Figure 6. The enrichment of Nb2O5 relative to TiO2 in the surface layer of the alloy can be explained with a preferential dissolution of the more reactive Ti during the exposure in piranha solution. This is a similar trend as it has been reported for example for pickling of Ti6Al7Nb.1999

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Figure 6. XPS peak area ratios of oxide/metal states for Ti40Nb samples with mechanically polished and chemically treated surface states (derived from spectra in Figure 5).

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The impact of the piranha treatment on the wettability was evaluated by means of contact angle measurements. Both the ground surface states show a partial wettability, however with a significant difference in value. The advancing contact angle yields 66 ± 1° for cp2-Ti and 56 ± 6° for Ti40Nb. The chemically treated surfaces show a total wettability with contact angles below 10° which can be explained by the changed nanotopography, in the first instance. Due to the spreading of the drops on cp2-Ti as well as on Ti40Nb it is not possible to gain distinct angle values. At least, chemically treated Ti40Nb tends toward lower contact angles than the respective cp2-Ti. This might be caused by the different surface chemistry established by XPS analysis, before.

In conclusion of these surface analytical studies, it can be stated that, though by the chemical treatment in H2SO4/H2O2 solution on the Ti40Nb alloy a surface nano-roughness very similar to that for cp2-Ti can be achieved, but the chemistry of the surface state is significantly different. Enhanced growth of the passive layer is observed in both cases. But while on cp-Ti mainly TiO2 forms,2007 the surface layer on Ti40Nb has a mixed oxide state with Nb-oxide being significantly enriched. As an additional benefit of this chemical treatment it should be mentioned that different to other acid etching treatments, for example, in HCl,2008 even longer exposure in this oxidative acidic solution does not lead to hydrogen absorption and hydride formation as it could be proven by hot gas extraction and XRD measurements (data not shown). Therefore, additional alteration of the subsurface with consequences for further surface treatments, mechanical properties and biological activity is not to be expected.

In vitro response of hMSC

For analyses of the hMSC response in vitro, sets of disc samples of the Ti40Nb alloy and of cp2-Ti with mechanically ground and chemically treated surface states, respectively, were employed. The tests were accomplished with fluorescence and electron microscopic imaging—selected micrographs are shown in Figure 7.

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Figure 7. Fluorescence and SEM micrographs of hMSC grown on mechanically ground and chemically treated surfaces of cp2-Ti and Ti40Nb after 2 and 24 h in culture (green fluorescence: F-actin, red fluorescence: vinculin, nuclei are stained in blue). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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The morphology of attached hMSC was visualized by fluorescence staining of cytoskeleton F-actin filaments and vinculin 2 h after seeding (Figure 7, top). The morphology of hMSC was not markedly changed by the different surfaces. The cells revealed a polygonal shape and showed slight alterations in the distribution pattern of F-actin filaments. hMSC on chemically treated surfaces showed after 2 h well organized F-actin fibers on both alloys; on cp2-Ti they spread much more. On mechanically ground surfaces a lot of F-actin monomers were located at the periphery; vinculin was distributed more cytosolic/perinuclear. SEM images after 2 h (Figure 7, middle) confirmed the results of immunofluorescence staining and showed more round cells on grounded material and more spread cells on chemically treated surfaces. After 24 h, hMSC became well spread on all materials and surfaces (Figure 7, bottom) with more filopodia on chemically treated surfaces than on grounded material.

Twenty-four hours after seeding, hMSC were analyzed for metabolic activity by MTS assay (Figure 8). MTS formazan formation was the highest on chemically treated Ti40Nb. The metabolic activity of hMSC on chemically treated cp2-Ti was only about 40% of that of hMSC on treated Ti40Nb. On the ground surfaces 36 and 60% less MTS formazan formation was found on Ti40Nb and cp2-Ti, respectively. It was described that metabolic activity correlates with DNA content/cell number,2012 so it can be concluded that Ti40Nb promotes not only the spreading but also the number of attached cells. On chemically treated surfaces always more cells were present in comparison to grounded material.

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Figure 8. Metabolic activity of hMSC after 24 hours – MTS assay; hMSC grown on mechanically ground and chemically treated surfaces of Ti40Nb and cp2-Ti; significant differences of Ti40Nb versus cp2-Ti are indicated with ***p < 0.001; n = 3.

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In the SEM, after 24 h almost all cells show a polygonal shape with large cytoplasmic extensions (Figure 7). On all surfaces an apparent alignment of the cells along the grinding grooves can be seen which indicates that such a kind of micro-roughness was recognized by the cells. Compared to the grounded samples, hMSC which were cultured on the chemically treated surfaces were clearly more spread after 24 h. Spreading along the grooves was more evident at the only ground sample surfaces. The accelerated adhesion and spreading of the cells on chemically treated samples, in particular on chemically treated Ti40Nb could cause the higher metabolic activity/cell number seen by MTS assay after 24 h. High resolution SEM (Figure 9) images of hMSCs on chemically treated Ti40Nb substrate show that this nanoscaled surface topography attracts filopodia and cytoplasmatic extensions. This also might be a reason for the accelerated adhesion of hMSC on the nano-roughened and oxidized surfaces compared to the respective ground ones.

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Figure 9. High resolution SEM micrograph of an extended filopodia (white arrows) on a chemically treated Ti40Nb surface after 24 h in culture.

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TNAP is an early differentiation marker of osteoblasts and osteogen differentiated hMSC.1998, 2004, 2008 In Figure 10, specific TNAP activity was the highest in cells on chemically treated Ti40Nb, followed by ground Ti40Nb, treated cp2-Ti, and ground cp2-Ti.

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Figure 10. Activity of tissue non-specific alkaline phosphatase (TNAP) after 11 days on mechanically ground and chemically treated surfaces of Ti40Nb and cp2-Ti; significant difference of Ti40Nb versus cp2-Ti is indicated with *p < 0.05; n = 3.

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SUMMARY AND CONCLUSIONS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. EXPERIMENTAL
  5. RESULTS AND DISCUSSION
  6. SUMMARY AND CONCLUSIONS
  7. Acknowledgments
  8. REFERENCES

The chemical treatment of surfaces of low modulus β-type Ti40Nb samples in H2SO4/H2O2 solution yields a characteristic nanoroughness which is related with a network of nearly semi-spherical nanopits of about 25 nm mean diameter. This nanotopography is very similar to that achieved for cp2-Ti, but the chemistry of the surface state is significantly different. While on cp2-Ti mainly TiO2 forms, already the native layer on a mechanically ground Ti40Nb surface has a mixed oxide state with Nb-oxides, mostly Nb2O5, contributing to the layer formation. The chemical treatment enhances in both cases the oxide layer growth. But for Ti40Nb this treatment causes also a strong enrichment of Nb2O5 relative to TiO2 in the surface layer. The in vitro analyses clearly indicated that chemically treated surfaces accelerate the adhesion and spreading of hMSC and increase the number of adherent cells. The enhanced TNAP activity at day 11 on chemically treated surfaces might be a consequence of the accelerated spreading. Those effects are significantly more pronounced for Ti40Nb than for cp2-Ti.

Surface topography and chemistry are decisive factors for the cell response to bone implant materials but their effects are usually hard to identify separately. Both influence the surface energy, hydrophilicity and presence of functional groups that determine the way of water and protein adsorption as first steps of the interactions between cells and implant surface. Cells are able to decipher those initial messages and accordingly, cell adhesion and proliferation are regulated.2010 Though control of implant surface states at every scale is recommended, recent studies clearly revealed that nanoscale surface modifications of metallic implant materials have a great potential to directly stimulate molecular and cellular reactions that take place at the same length scale.2006, 2004 In recent works, the Nanci group demonstrated that nanoscale oxidative patterning of Ti by chemical treatments in H2SO4/H2O2 solutions has beneficial effects on both initial and osteogenic events in vitro. However, they also emphasized that the observed effects are hard to explain since the underlying processes are very complex.2006, 2010, 1998

In this study, we could confirm those positive influences of chemically treated nano-rough surfaces on cell adhesion and spreading. This could arise from the markedly improved wettability on the one hand. On the other hand, this might be related to the morphology of the chemically treated surfaces. They also show the tendency to promote osteogenic differentiation as seen with TNAP activity at day 11. In comparison of the materials, adhesion and differentiation of hMSC was always accelerated on Ti40Nb alloy. Since the established micro- and nanotopographies were very similar for both materials surfaces, the changes in the surface chemistry and therefore the wettability appear to be most decisive. The presence and dominance of Nb-oxide species in the surface layer has certainly a strong influence on the surface energy and surface charge state of the alloy. While for commercially used cp2-Ti those aspects and their consequences for surface wettability and cell biological responses are already quite well studied,2009, 2006, 2010, 2009 for Nb or Nb-containing alloys only a few and in part controversial findings are reported.2009, 2006 As a matter of fact, a change in wettability depending on the nature of the dominating transition metal oxide on the material surfaces must be considered.1998 Moreover, the incorporation of Nb-oxide species in the oxide layer on the Ti40Nb alloy strengthens its barrier-type nature and thus, reduces the corrosion rate.2006, 2013 On one hand, this relates to reduced metal ion release rates, but also the counter reaction, that is, the reduction of oxygen which is dissolved in the physiological solution can be markedly suppressed. Comprehensive studies on cp-Ti and on Ti6Al4V, for example, by Kalbacova et al.,2007 revealed the detrimental effect of reactive oxygen species (radicals and hydrogen peroxide), which are intermediate products of the complex oxygen reduction reaction, on the metabolic activity of osteoblasts and monocytes/macrophages. In perspective, similar studies with Ti–Nb alloys have to be conducted. Furthermore, the transfer of the positive effects of chemically treated Ti40Nb observed in in vivo tests to in vivo conditions must be critically assessed.

Acknowledgments

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. EXPERIMENTAL
  5. RESULTS AND DISCUSSION
  6. SUMMARY AND CONCLUSIONS
  7. Acknowledgments
  8. REFERENCES

The authors are grateful to M. Frey, S. Donath, H. Bußkamp, M. Johne, B. Opitz, and C. Preissler for technical assistance. The authors thank Prof. M. Bornhäuser for providing the hMSC. M. Uhlemann, A. Teresiak, and H. Worch are acknowledged for fruitful discussions.

REFERENCES

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  3. INTRODUCTION
  4. EXPERIMENTAL
  5. RESULTS AND DISCUSSION
  6. SUMMARY AND CONCLUSIONS
  7. Acknowledgments
  8. REFERENCES
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